Assessing renal structural alterations and outcomes

ABSTRACT

This document provides methods and materials involved in assessing renal structural alterations (e.g., renal fibrosis, glomerular basement thickening, mesangial matrix expansion, swollen podocytes, and foot processes effacement) as well as methods and materials involved in assessing outcomes. For example, methods and materials for using the level of urinary CNP (e.g., a urinary to plasma CNP ratio) to determine whether or not a mammal is developing renal structural alterations (e.g., renal fibrosis, glomerular basement thickening, mesangial matrix expansion, swollen podocytes, and foot processes effacement) as well as methods and materials for using the level of urinary CNP levels to identify patients having an increased likelihood of experiencing a poor outcome are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/580,139, filed Dec. 23, 2011. The disclosure of the priorapplication is considered part of (and is incorporated by reference in)the disclosure of this application.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grants R01-HL36634and P01-HL7661 awarded by National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in assessingrenal structural alterations (e.g., renal fibrosis) as well as methodsand materials involved in assessing outcomes. For example, this documentrelates to methods and materials for using the level of urinary C-typenatriuretic peptide (CNP) (e.g., a urinary to plasma CNP ratio) or thelevel of plasma CNP to determine whether or not a mammal is developingrenal structural alterations (e.g., renal fibrosis, glomerular basementthickening, swollen podocytes, and foot processes effacement). Thisdocument also relates to methods and materials for using the level ofurinary or plasma CNP, which can include six molecular CNP forms, levelsto identify heart failure patients having an increased likelihood ofexperiencing a poor outcome and who may have disease processes known toinvolve the kidney including, but not limited to, heart failure,hypertension, diabetes, metabolic syndrome, and chronic kidney disease

2. Background Information

C-type natriuretic peptide (CNP) is part of the natriuretic peptidefamily, produced in the kidney as well as the endothelium and can bedetected in the plasma and urine. It is synthesized as the precursor 103amino acid (AA) protein, proCNP (AA 1-103), which is then cleaved intoCNP-53 (AA 51-103) and NT-proCNP (AA 1-50) by the intracellularendoprotease furin. Additional downstream processing, by an unknownenzyme, cleaves CNP-53 to give rise to the primary biologically activeform CNP-22 (AA 82-103) and its amino-terminal, NT-CNP-53 (51-81).

CNP possesses potent anti-fibrotic and anti-proliferative propertiesthrough the activation of the natriuretic peptide receptor B (NPR-B),otherwise known as guanylyl cyclase receptor B (GC-B), and thegeneration of the second messenger 3′,5′-cyclic guanosine monophosphate(cGMP). CNP has limited natriuretic and diuretic actions.

SUMMARY

This document provides methods and materials involved in assessing renalstructural alterations (e.g., renal fibrosis, glomerular basementthickening, swollen podocytes, and foot processes effacement). Forexample, this document provides methods and materials for using thelevel of urinary CNP (e.g., a urinary to plasma CNP ratio) and/or thelevel of plasma CNP to determine whether or not a mammal is developingor is likely to develop renal structural alterations (e.g., renalfibrosis, glomerular basement thickening, mesangial matrix expansion,swollen podocytes, and foot processes effacement). Determining if ahuman patient is developing or is likely to develop renal structuralalterations by assessing the level of urinary or plasma CNP (e.g., aurinary to plasma CNP ratio) can aid in the identification of humanswith preclinical renal structural changes prior to the onset of symptomsand disease, thereby allowing for the initiation of strategies designedto prevent the progression of chronic kidney disease.

This document also provides methods and materials involved in assessingheart failure outcomes. For example, this document provides methods andmaterials for using the level of urinary or plasma CNP and/or its sixpossible molecular forms (FIG. 8) to identify humans having an increasedlikelihood of experiencing a poor outcome. Identify patients as havingan increased likelihood of experiencing a poor outcome based at least inpart on an elevated level of urinary CNP and/or a reduced level ofplasma CNP can aid physicians and patients in making proper treatmentdecisions.

In general, one aspect of this document features a method for assessingrenal structure. The method comprises, or consist essentially of,determining whether or not a mammal contains an elevated urinary CNP toplasma CNP ratio, wherein the presence of the elevated urinary CNP toplasma CNP ratio indicates that the mammal contains or is likely toexperience renal structural alterations, and wherein the absence of theelevated urinary CNP to plasma CNP ratio indicates that the mammal doesnot contain and is not likely to experience the renal structuralalterations. The mammal can be a human. The renal structural alterationscan include renal fibrosis. The elevated urinary CNP to plasma CNP ratiocan be greater than 12,000 pg/day to 16 pg/mL. The elevated urinary CNPto plasma CNP ratio can be greater than 13,200 pg/day to 18 pg/mL.

In another aspect, this document features a method for assessing renalstructure. The method comprises, or consists essentially of, determiningwhether or not a mammal contains an elevated urinary CNP-22 to plasmaCNP-22 ratio, wherein the presence of the elevated urinary CNP-22 toplasma CNP-22 ratio indicates that the mammal contains or is likely toexperience renal structural alterations, and wherein the absence of theelevated urinary CNP-22 to plasma CNP-22 ratio indicates that the mammaldoes not contain and is not likely to experience the renal structuralalterations. The mammal can be a human. The renal structural alterationscan include renal fibrosis. The elevated urinary CNP-22 to plasma CNP-22ratio can be greater than 12,000 pg/day to 16 pg/mL. The elevatedurinary CNP-22 to plasma CNP-22 ratio can be greater than 13,200 pg/dayto 18 pg/mL.

In another aspect, this document features a method for assessing renalstructure. The method comprises, or consists essentially of, (a)detecting the presence of an elevated urinary CNP to plasma CNP ratio ina mammal, and (b) classifying the mammal as having or as likely toexperience a renal structural alteration based at least in part on thepresence. The mammal can be a human. The renal structural alteration caninclude renal fibrosis. The elevated urinary CNP to plasma CNP ratio canbe greater than 12,000 pg/day to 16 pg/mL. The elevated urinary CNP toplasma CNP ratio can be greater than 13,200 pg/day to 18 pg/mL.

In another aspect, this document features a method for assessing renalstructure. The method comprises, or consists essentially of, (a)detecting the presence of an elevated urinary CNP-22 to plasma CNP-22ratio in a mammal, and (b) classifying the mammal as having or as likelyto experience a renal structural alteration based at least in part onthe presence. The mammal can be a human. The renal structural alterationcan include renal fibrosis. The elevated urinary CNP-22 to plasma CNP-22ratio can be greater than 12,000 pg/day to 16 pg/mL. The elevatedurinary CNP-22 to plasma CNP-22 ratio can be greater than 13,200 pg/dayto 18 pg/mL.

In another aspect, this document features a method for assessingoutcomes. The method comprises, or consists essentially of, determiningwhether or not a mammal having experienced a disease process contains anelevated level of urinary CNP, wherein the presence of the elevatedlevel indicates that the mammal is likely to experience a poor outcome,and wherein the absence of the level indicates that the mammal is notlikely to experience the poor outcome. The mammal can be a human. Thedisease process can be heart failure, hypertension, diabetes, metabolicsyndrome, or chronic kidney disease. The poor outcome can be death,hospitalization, heart failure, myocardial infarction, worsening renalfunction, worsening cardiac function, or dialysis. The urinary CNP canbe urinary NT-CNP-53. The elevated level can be greater than 36,000 pgof NT-CNP-53/day. The elevated level can be greater than 42,000 pg ofNT-CNP-53/day.

In another aspect, this document features a method for assessingoutcomes. The method comprises, or consists essentially of, (a)detecting the presence of an elevated urinary CNP in a mammal havingexperienced a disease process, and (b) classifying the mammal as likelyto experience a poor outcome based at least in part on the presence. Themammal can be a human. The disease process can be heart failure,hypertension, diabetes, metabolic syndrome, or chronic kidney disease.The poor outcome can be death, hospitalization, heart failure,myocardial infarction, worsening renal function, worsening cardiacfunction, or dialysis. The urinary CNP can be urinary NT-CNP-53. Theelevated level can be greater than 36,000 pg of NT-CNP-53/day. Theelevated level can be greater than 42,000 pg of NT-CNP-53/day.

In another aspect, this document features a method for assessing renalstructure. The method comprises, or consists essentially of, (a)performing an immunoassay with an anti-CNP antibody to detect anelevated urinary CNP to plasma CNP ratio of a mammal, and (b)classifying the mammal as containing or as likely to experience renalstructural alterations. The mammal can be a human. The renal structuralalterations can comprise renal fibrosis. The elevated urinary CNP toplasma CNP ratio can be greater than 12,000 pg/day to 16 pg/mL. Theelevated urinary CNP to plasma CNP ratio can be greater than 13,200pg/day to 18 pg/mL.

In another aspect, this document features a method for assessing renalstructure. The method comprises, or consists essentially of, (a)performing an immunoassay with an anti-CNP antibody to detect anelevated urinary CNP-22 to plasma CNP-22 ratio of a mammal, and (b)classifying the mammal as containing or as likely to experience renalstructural alterations. The mammal can be a human. The renal structuralalterations can comprise renal fibrosis. The elevated urinary CNP-22 toplasma CNP-22 ratio can be greater than 12,000 pg/day to 16 pg/mL. Theelevated urinary CNP-22 to plasma CNP-22 ratio can be greater than13,200 pg/day to 18 pg/mL.

In another aspect, this document features a method for assessingoutcomes. The method comprises, or consists essentially of, (a)performing an immunoassay with an anti-CNP antibody to detect thepresence of an elevated level of urinary CNP in a mammal havingexperienced a disease process, and (b) classifying the mammal as likelyto experience a poor outcome based at least in part on the presence. Themammal can be a human. The disease process can be heart failure,hypertension, diabetes, metabolic syndrome, or chronic kidney disease.The poor outcome can be death, hospitalization, heart failure,myocardial infarction, worsening renal function, worsening cardiacfunction, or dialysis. The urinary CNP can be urinary NT-CNP53. Theelevated level can be greater than 36,000 pg of NT-CNP53/day. Theelevated level can be greater than 42,000 pg of NT-CNP53/day.

In another aspect, this document features a method for assessing amammal for an increased risk of death or myocardial infarction. Themethod comprises, or consists essentially of, determining whether or notthe mammal contains an elevated level of plasma CNP-22, wherein thepresence of the elevated level indicates that the mammal is likely toexperience the death or myocardial infarction sooner than a comparablemammal lacking the elevated level, and wherein the absence of theelevated level indicates that the mammal is likely to experience thedeath or myocardial infarction later than a comparable mammal having theelevated level. The mammal can be a human. The elevated level can begreater than 14 pg/mL. The elevated level can be greater than 16 pg/mL.

In another aspect, this document features a method for assessing amammal for an increased risk of death or myocardial infarction. Themethod comprises, or consists essentially of, (a) performing animmunoassay with an anti-CNP antibody to detect the presence of anelevated level of plasma CNP-22 in a mammal, and (b) classifying themammal as likely to experience death or myocardial infarction based atleast in part on the presence. The mammal can be a human. The elevatedlevel can be greater than 14 pg/mL. The elevated level can be greaterthan 16 pg/mL. The method can comprise classifying the mammal as likelyto experience death or myocardial infarction sooner than a comparablemammal lacking the elevated level.

In another aspect, this document features a method for treating a mammalhaving an increased risk of a renal structural alteration. The methodcomprises, or consists essentially of, (a) determining that the mammalhas an elevated urinary CNP to plasma CNP ratio, (b) monitoring themammal for the presence of a risk factor for renal structuralalteration, and (c) instructing the mammal to administer a therapeuticagent to reduce a symptom of the renal structural alteration. The mammalcan be a human. The elevated urinary CNP to plasma CNP ratio can begreater than 12,000 pg/day to 16 pg/mL. The elevated urinary CNP toplasma CNP ratio can be greater than 13,200 pg/day to 18 pg/mL. The riskfactor can be selected from the group consisting of an age factor,hypertension, an elevated serum creatinine level, proteinuria, anelevated body mass index, an elevated cholesterol level, a smokinghabit, and diabetes. The therapeutic agent can be an ACE inhibitor, anangiotensin receptor blocker, an aldosterone antagonist, a statin, anative natriuretic peptide, or a designer natriuretic peptide.

In another aspect, this document features a method for treating a mammalhaving an increased risk of a poor outcome. The method comprises, orconsists essentially of, (a) determining that the mammal has an elevatedlevel of urinary CNP, (b) monitoring the mammal for the presence of arisk factor for a poor outcome, and (c) instructing the mammal toadminister a therapeutic agent to reduce the likelihood of the pooroutcome. The mammal can be a human. The urinary CNP can be urinaryNT-CNP-53. The elevated level can be greater than 36,000 pg ofNT-CNP-53/day. The elevated level can be greater than 42,000 pg ofNT-CNP-53/day. The risk factor can be selected from the group consistingof an age factor, hypertension, an elevated serum creatinine level,proteinuria, an elevated body mass index, an elevated cholesterol level,a smoking habit, and diabetes. The therapeutic agent can be an ACEinhibitor, an angiotensin receptor blocker, an aldosterone antagonist, astatin, a native natriuretic peptide, or a designer natriuretic peptide.

In another aspect, this document features a method for treating a mammalhaving an increased risk of myocardial infarction. The method comprises,or consists essentially of, (a) determining that the mammal has anelevated level of plasma CNP-22, (b) monitoring the mammal for thepresence of a risk factor for myocardial infarction, and (c) instructingthe mammal to administer a therapeutic agent to reduce the risk ofmyocardial infarction. The mammal can be a human. The elevated level canbe greater than 14 pg/mL. The elevated level can be greater than 16pg/mL. The risk factor can be selected from the group consisting of anage factor, hypertension, an elevated serum creatinine level,proteinuria, an elevated body mass index, an elevated cholesterol level,a smoking habit, and diabetes. The therapeutic agent can be an ACEinhibitor, an angiotensin receptor blocker, an aldosterone antagonist, astatin, a native natriuretic peptide, or a designer natriuretic peptide.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1. Representative histology images at 20× objective magnification(A) and quantification of picrosirius red staining (B) of renal corticalfibrosis from 2, 11, and 20 month old Fischer rats. Values are mean±SE.n=7 for all age groups. *P<0.05 vs. 2 months, † P<0.05 vs. 11 months.

FIG. 2. Representative histology images at 20× objective magnification(A) and quantification of picrosirius red staining (B) of renalmedullary fibrosis from 2, 11, and 20 month old Fischer rats. Values aremean±SE. n=7 for all age groups. *P<0.05 vs. 2 months, † P<0.05 vs. 11months.

FIG. 3. Representative electron micrographs at 8000× magnification ofthe glomerulus from 2 (A), 11 (B), and 20 (C) month old Fischer rats.

FIG. 4. Quantification of glomerular basement thickness from 2, 11, and20 month old Fischer rats. Values are mean±SE. n=5 for all age groups.*P<0.05 vs. 2 months, † P<0.05 vs. 11 months.

FIG. 5. Representative images at 20× objective magnification of theimmunohistochemical localization of renal cortical and medullary CNPfrom 2, 11, and 20 month old Fischer rats.

FIG. 6. Changes in plasma CNP (A), urinary CNP excretion (B), urinary toplasma CNP ratio (C), and proteinuria (D) between 2, 11, and 20 monthold Fischer rats. Values are mean±SE. n=10 for all age groups. *P<0.05vs. 2 months, † P<0.05 vs. 11 months.

FIG. 7. Correlations between urinary CNP excretion and renal cortical(A) and medullary (B) fibrosis, as well as between urinary to plasma CNPratio and renal cortical (C) and medullary (D) fibrosis.

FIG. 8 is a diagram of the molecular forms of CNP.

FIG. 9 is a listing of the amino acid sequences of the molecular formsof CNP.

FIG. 10. CNP immunohistochemical localization in renal tissue from younghuman donors (A-C) and old human donors (D-F) at 20× objectivemagnification. Negative control (G).

FIG. 11. Correlations between glomerular basement membrane (GBM)thickness and urinary CNP excretion (A) as well as between GBM thicknessand urinary to plasma CNP (B).

FIG. 12. Urinary KIM-1 excretion in ADHF patients (HHF) vs. controls.Outlier box plots displayed with median and interquartile ranges (IQR;box) and 1.5*IQR (error bars) for 24 h KIM-1 excretion values.

FIGS. 13A-C. Association between NT-CNP-53 excretion and clinicaloutcomes: (A) mortality, (B) time to first non-elective (all-cause)rehospitalization/death, (C) time to first non-elective cardiovascularrehospitalization/death. Outlier box plots displayed with median andinterquartile ranges (IQR; box) and 1.5*IQR (error bars) for 24 hNT-CNP-53 excretion values (natural logarithmic transformed data).

FIGS. 14A-C. Urinary excretion of CNP molecular forms by primary outcome(mortality) in ADHF. Outlier box plots displayed with median andinterquartile ranges (IQR; box) and 1.5*IQR (error bars) for 24 hoururinary (A) CNP22, (B) CNP53, and (C) NT-CNP53 excretion values againstmortality in ADHF.

FIGS. 15A-B. Kaplan-Meier curves for death (A) and myocardial infarction(MI; B) in the general population according to quartiles of plasmaCNP-22 levels. CNP-22 Q1 is from 2.0 to 10.1 pg/mL; CNP-22 Q2 is from10.2 to 13.1 pg/mL; CNP-22 Q3 is from 13.2 to 16.7 pg/mL; and CNP-22 Q4is from 16.8 to 265.0 pg/mL.

DETAILED DESCRIPTION

This document provides methods and materials involved in assessing renalstructural alterations (e.g., renal fibrosis). For example, thisdocument provides methods and materials for using the level of urinaryCNP (e.g., a urinary to plasma CNP ratio) and/or the level of plasma CNPto determine whether or not a mammal is developing or is likely todevelop a renal structural alteration. As described herein, the presenceof an elevated level of urinary CNP, a reduced level of plasma CNP,and/or an elevated level of a urinary to plasma CNP ratio can indicatethat the mammal is developing or is likely to develop a renal structuralalteration. Examples of renal structural alteration include, withoutlimitation, renal fibrosis, glomerular basement membrane thickening,mesangial matrix expansion, swollen podocytes, and foot processeseffacement. The methods and materials provided herein can be used toassess renal structural alterations in any appropriate mammal including,without limitation, humans, monkeys, horses, cows, sheep, goats, mice,and rats.

The amino acid sequences of six molecular forms of human CNP are setforth in FIGS. 8 and 9.

The term “elevated level” as used herein with respect to the plasma orurinary level of CNP (or a particular molecular form of CNP such asCNP-53) refers to any level that is above a median plasma or urinarylevel for an age-matched random population of healthy mammals (e.g., anage-matched random population of 10, 20, 30, 40, 50, 100, or 500 healthymammals) that do not have renal disease. In some cases, an elevatedlevel of plasma CNP (e.g., plasma CNP-22) can be any level that isgreater than 16 pg/mL. In some cases, an elevated level of urinary CNP(e.g., urinary CNP-22) can be any level that is greater than 12,000pg/day.

The term “elevated” as used herein with respect to a urinary CNP toplasma CNP ratio refers to any ratio level that is above an averageurinary CNP to plasma CNP ratio for an age-matched random population ofhealthy mammals (e.g., an age-matched random population of 10, 20, 30,40, 50, 100, or 500 healthy mammals) that do not have renal disease. Insome cases, an elevated urinary CNP to plasma CNP ratio can be a ratiothat is greater than 12,000 pg/day urinary CNP to 16 pg/mL plasma CNP.In some cases, a plasma CNP to urinary CNP ratio can be used in place ofa urinary CNP to plasma CNP ratio.

In some cases, the presence of a reduced level of plasma CNP canindicate that the mammal is developing or is likely to develop a renalstructural alteration or is likely to experience a poor outcome. Theterm “reduced level” as used herein with respect to the plasma level ofCNP refers to any level that is below a median plasma level for anage-matched random population of healthy mammals (e.g., an age-matchedrandom population of 10, 20, 30, 40, 50, 100, or 500 healthy mammals)that do not have renal disease. In some cases, a reduced level of plasmaCNP can be any level that is less than 10 pg/mL.

In some cases, the presence of a reduced level of plasma CNP, anelevated level of urinary CNP, and an elevated level of plasma NT-proBNPcan indicate that the mammal is developing or is likely to develop arenal structural alteration or is likely to experience a poor outcome.The term “elevated level” as used herein with respect to the plasmalevel of NT-proBNP refers to any level that is above a median plasmalevel for an age-matched random population of healthy mammals (e.g., anage-matched random population of 10, 20, 30, 40, 50, 100, or 500 healthymammals) that do not have renal disease. In some cases, an elevatedlevel of plasma NT-proBNP can be any level that is greater than 450pg/mL.

Any appropriate method can be used to determine a urinary CNP level, aplasma CNP level, a urinary CNP to plasma CNP ratio, a plasma NT-proBNPlevel, or a plasma CNP to urinary CNP ratio. For example, polypeptidedetection methods such as immunoassays (e.g., ELISAs orradioimmunoassays) and mass spectrometry can be used to determine thelevel of CNP in a plasma or urine sample. In some cases,radioimmunoassays can be used to determine the urinary CNP to plasma CNPratio.

This document also provides methods and materials involved in assessingoutcomes. For example, this document provides methods and materials forusing the level of urinary CNP or its six possible molecular forms(e.g., CNP-53 or NT-CNP-53) to determine whether or not a mammal islikely to experience a poor outcome. As described herein, the presenceof an elevated level of urinary CNP can indicate that the mammal islikely to experience a poor outcome. Examples of poor outcomes include,without limitation, death, hospitalization, heart failure, myocardialinfarction, worsening renal function, worsening cardiac function, anddialysis. The methods and materials provided herein can be used toassess outcomes in any appropriate mammal including, without limitation,humans, monkeys, horses, cows, sheep, and goats.

In some cases, the term “elevated level” as used herein with respect tothe urinary level of NT-CNP-53 can refer to any level that is above amedian urinary NT-CNP-53 level for an age-matched random population ofhealthy mammals (e.g., an age-matched random population of 10, 20, 30,40, 50, 100, or 500 healthy mammals) that do not have a history of otherco-morbidities such as heart failure, hypertension, diabetes, metabolicsyndrome, or chronic kidney disease. In some cases, an elevated level ofurinary NT-CNP-53 can be any level that is greater than 36,000 pg/day.

This document also provides methods and materials to assist medical orresearch professionals in determining whether or not a mammal isdeveloping or is likely to develop a renal structural alteration as wellas methods and materials to assist medical or research professionals indetermining whether or not a mammal is likely to experience a pooroutcome. Medical professionals can be, for example, doctors, nurses,medical laboratory technologists, and pharmacists. Researchprofessionals can be, for example, principal investigators, researchtechnicians, postdoctoral trainees, and graduate students. Aprofessional can be assisted in determining whether or not a mammal isdeveloping or is likely to develop a renal structural alteration by (1)determining a urinary CNP level, a plasma CNP level, a urinary CNP toplasma CNP ratio, or a plasma CNP to urinary CNP ratio, and (2)communicating information about that level or ratio to thatprofessional. A professional can be assisted in determining whether ornot a mammal is likely to experience a poor outcome by (1) determining aurinary CNP level, and (2) communicating information about that level tothat professional.

Any method can be used to communicate information to another person(e.g., a professional). For example, information can be given directlyor indirectly to a professional. In addition, any type of communicationcan be used to communicate the information. For example, mail, e-mail,telephone, and face-to-face interactions can be used. The informationalso can be communicated to a professional by making that informationelectronically available to the professional. For example, theinformation can be communicated to a professional by placing theinformation on a computer database such that the professional can accessthe information. In addition, the information can be communicated to ahospital, clinic, or research facility serving as an agent for theprofessional.

This document also provides methods and materials for treating a mammalthat is developing or is likely to develop a renal structuralalteration. For example, a mammal can be assessed to determine if themammal has an elevated urinary CNP to plasma CNP ratio. As describedherein, mammals having an elevated urinary CNP to plasma CNP ratio canbe developing or can be likely to develop a renal structural alteration.Once a mammal is identified as having an elevated urinary CNP to plasmaCNP ratio, that mammal can be monitored for the presence of one or morerisk factors for a renal structural alteration. For example, the mammalcan be monitored or evaluated for the presence of an age factor,hypertension, an elevated serum creatinine level, proteinuria, a malegender, an elevated body mass index, an elevated cholesterol level, asmoking habit, and/or diabetes. Once a mammal having an elevated urinaryCNP to plasma CNP ratio and one or more risk factors is identified, thatmammal can be treated with one or more therapeutic agents designed toreduce or counter-act a symptom of a renal structural alteration. Forexample, a mammal having an elevated urinary CNP to plasma CNP ratio andhypertension can be treated with an ACE inhibitor, an angiotensinreceptor blocker (ARB), an aldosterone antagonist, a statin, a nativenatriuretic peptide, or a designer natriuretic peptide to reduce asymptom of a renal structural alteration. In some cases, a mammal havingan elevated urinary CNP to plasma CNP ratio and one or more risk factorscan be instructed to self-treat with one or more therapeutic agentsdesigned to reduce or counter-act a symptom of a renal structuralalteration.

This document also provides methods and materials for treating a mammalthat is likely to experience a poor outcome (e.g., death,hospitalization, heart failure, myocardial infarction, worsening renalfunction, worsening cardiac function, and dialysis). For example, amammal can be assessed to determine if the mammal has an elevated levelof urinary CNP or its six possible molecular forms (e.g., CNP-53 orNT-CNP-53). As described herein, mammals having an elevated level ofurinary CNP or its six possible molecular forms (e.g., CNP-53 orNT-CNP-53) can be likely to experience a poor outcome. Once a mammal isidentified as having an elevated level of urinary CNP or its sixpossible molecular forms (e.g., CNP-53 or NT-CNP-53), that mammal can bemonitored for the presence of one or more risk factors of a pooroutcome. For example, the mammal can be monitored or evaluated for thepresence of an age factor, hypertension, an elevated serum creatininelevel, proteinuria, a male gender, an elevated body mass index, anelevated cholesterol level, a smoking habit, and/or diabetes. Once amammal having an elevated level of urinary CNP or its six possiblemolecular forms (e.g., CNP-53 or NT-CNP-53) and one or more risk factorsis identified, that mammal can be treated with one or more therapeuticagents designed to reduce the likelihood of a poor outcome. For example,a mammal having an elevated level of urinary CNP or its six possiblemolecular forms (e.g., CNP-53 or NT-CNP-53) and hypertension can betreated with an ACE inhibitor, an ARB, an aldosterone antagonist, astatin, a native natriuretic peptide, or a designer natriuretic peptideto reduce the likelihood of a poor outcome. In some cases, a mammalhaving an elevated level of urinary CNP or its six possible molecularforms (e.g., CNP-53 or NT-CNP-53) and one or more risk factors can beinstructed to self-treat with one or more therapeutic agents designed toreduce the likelihood of a poor outcome.

This document also provides methods and materials for treating mammals.For example, a mammal can be assessed to determine if the mammal has anelevated level of plasma CNP-22. As described herein, mammals having anelevated level of plasma CNP-22 can have an increased risk for death ormyocardial infarction. Once a mammal is identified as having an elevatedlevel of plasma CNP-22, that mammal can be monitored for the presence ofone or more risk factors for death or myocardial infarction. Forexample, the mammal can be monitored or evaluated for the presence of anage factor, hypertension, an elevated serum creatinine level,proteinuria, a male gender, an elevated body mass index, an elevatedcholesterol level, a smoking habit, and/or diabetes. Once a mammalhaving an elevated level of plasma CNP-22 and one or more risk factorsis identified, that mammal can be treated with one or more therapeuticagents designed to reduce the mammal's risk of suffering from death ormyocardial infarction. For example, a mammal having an elevated level ofplasma CNP-22 and an elevated cholesterol level can be treated with anACE inhibitor, an ARB, an aldosterone antagonist, a statin, a nativenatriuretic peptide, or a designer natriuretic peptide to reduce themammal's risk of suffering from death or myocardial infarction. In somecases, a mammal having an elevated level of plasma CNP-22 and one ormore risk factors can be instructed to self-treat with one or moretherapeutic agents designed to reduce the mammal's risk of sufferingfrom death or myocardial infarction.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Use of Urinary CNP Excretion as Biomarker for RenalFibrosis During Aging Animals

Studies were performed in 2, 11, and 20 month old male Fischer rats(Harlan Laboratories, Inc., Madison, Wis., n=8-10 per age group, unlessotherwise specified). The experimental study was performed in accordancewith the Animal Welfare Act and with approval of the Mayo ClinicInstitutional Animal Care and Use Committee.

Human Renal Biopsy Tissue

Human kidney tissue was obtained from core needle biopsy specimens fromhealthy living kidney donors at the time of kidney donation as describedelsewhere (Rule et al., Ann. Intern. Med., 152:561-567 (2010)). A totalof six paraffin-embedded renal biopsy specimens were examined in thisstudy. The young group consisted of three female donors with mean age of19 years old (age range 18 to 20 years old) and old group consisted ofthree 71 year old female donors.

24 Hours Urine Collection

Rats were placed in metabolic cages with free access to food and waterand acclimatized for 24 hours. Following the acclimatizing period, urinewas then collected for 24 hours for proteinuria and CNP assessment.Urinary protein excretion was measured on 24 hour urine samples usingthe Pyrogallol Red dye-binding assay.

Acute Studies for Blood Pressure, Glomerular Filtration Rate, and PlasmaCollection

Rats were anesthetized (1.5% isoflurane in oxygen), and PE-50 tubing wasplaced into the carotid artery for blood pressure (BP) acquisition usingCardioSOFT Pro software (Sonometrics Corporation, London, Ontario) andblood sampling. The bladder was cannulated for urine collection. Thejugular vein was cannulated with PE-50 tubing and was continuouslyinfused with 2% inulin (Sigma, St. Louis, Mo.) in normal saline. After60 minutes of equilibration, a clearance study was performed. Theclearance study lasted 60 minutes, and urine was collected with bloodsampling at the end of the clearance study to calculate GFR from theclearance of inulin and for measuring plasma CNP. Blood was collectedfrom the carotid artery and placed in EDTA tubes on ice (Stingo et al.,Am. J. Phys., 263:H1318-1321 (1992)). Blood was immediately centrifugedat 2,500 rpm at 4° C. for 10 minutes, and the plasma was stored inpolystyrene tubes at −80° C. for future use. Inulin concentrations weremeasured using the anthrone method for GFR analysis as describedelsewhere (Davidson and Sackner, J. Lab. Clin. Med., 62:351-356 (1963)).

Rat Renal Tissue

After the acute study, the rat kidneys were removed for total weightsand were then divided into sections. A cross-section of the renal tissuewas preserved in 10% formalin for histological analysis of fibrosis andCNP, and smaller cube sections were preserved in 2.5% glutaraldehyde forelectron microscopy (EM) analysis.

Histological Analysis of Fibrosis

Fixed rat renal tissues (n=7) were dehydrated, embedded in paraffin andsectioned at thickness of 4 μm. Collagen and extend of fibrosis wasperformed with picrosirius red staining. An Axioplan II KS 400microscope (Carl Zeiss, Inc., Germany) was used to capture at least 4randomly selected images from each slide using a 20× objective and KS400 software was utilized to determined fibrotic area as a percentage oftotal tissue area.

Electron Microscopic Analysis

Rat renal tissues fixed in 2.5% glutaraldehyde were dehydrated andembedded in a resin mould. Ultra-thin sections were cut according to theEM core facility procedures. The glomeruli were imaged at 5000× and8000× magnifications using a JEM-1400 transmission electron microscope.

Glomerular Basement Membrane Thickness Measurements

Glomerular EM images were captured at 5000× magnification from each agegroup (n=5) of rats. The thicknesses of the GBM were measured using theapplication Digital Micrograph (Gatan Inc., Pleasanton, Calif.). Foreach rat, 20 measurements were performed by an experienced EM technicianand the data were subjected to an Excel morphometrics macro giving themean thickness in nanometers (nm).

Plasma and Urinary CNP

Plasma and urinary CNP-22 was determined as described elsewhere (Stingoet al., Am. J. Phys., 263:H1318-1321 (1992)) using commerciallyavailable non-equilibrium radioimmunoassay kits from PhoenixPharmaceutical (Mountain View, Calif.) and an antibody to human CNP-22,which is fully cross-reactive to rat CNP-22. One mL of plasma wasextracted using C-18 Bond Elut cartridges. After washing cartridges with4 mL 100% methanol and 4 mL water, plasma was applied, and thecartridges were washed. Eluates were concentrated on a Savant speedvacuum concentrator, and pellets were re-suspended in 300 μL of assaybuffer. 100 μL of standards and samples were incubated with 100 μL ofanti-human CNP at 4° C. After 18 hours, 100 μL (10,000 counts) ofI¹²⁵-labeled CNP was added and incubated at 4° C. for 18 hours. Then, asecond antibody was added to all samples to separate the free and boundfractions, and the samples were centrifuged. The free fraction wasaspirated, and the bound fraction was counted on a gamma counter. Astandard curve was generated and used to calculate the concentrations ofthe unknown samples, which were reported in pg/mL. The range of thestandard curve was 0.5 to 128 pg, with a lower limit of detection of 0.5pg. Inter- and intra-assay variability was 11% and 5.2%, respectively.Recovery was 72±6%. Cross-reactivity was <1% with ANP, BNP, endothelin,and adrenomedullin, and 97% with CNP-53.

CNP Immmunohistochemistry

The presence of renal CNP immunoreactivity was assessed as describedelsewhere (Stingo et al., Am. J. Phys., 263:H1318-1321 (1992)). Briefly,slides with paraffin-embedded renal tissues were incubated in a 60° C.oven for 2 hours and then deparaffinized using established laboratoryprocedures. After deparaffinization, slides were incubated with 0.6%hydrogen peroxide in methanol for 20 minutes at room temperature toblock endogenous peroxidase activity, and then 5% normal goat serum wasused to block nonspecific protein binding sites before primary antibodywas applied. Sections were placed in a moist chamber for 18-24 hours atroom temperature with the primary antibody (rabbit anti-human CNP-22,Phoenix Pharmaceutical, Mountain View, Calif.) at a dilution of 1:500.Control slides were treated with normal rabbit serum. Sections wereincubated with goat anti-rabbit IgG covalently linked to horseradishperoxidase and 3-amino-9-ethyl-carbazole substrate for peroxidasevisualization and were counterstained with hematoxylin to enhancenuclear detail. Staining slides were then viewed and interpreted by arenal pathologist blinded to the age groups.

Statistical Analysis

Results were expressed as mean±SE. Comparisons within groups were madeby ANOVA followed by Newman-Keuls multiple comparison test. A Pearsoncorrelation was performed to calculate the correlation between urinaryCNP excretion and renal fibrosis as well as GFR and between the urinaryto plasma CNP ratio and renal fibrosis. GraphPad Prism software(GraphPad Software, La Jolla, Calif.) was used for the abovecalculations. Statistical significance was accepted as P<0.05.

Results

Anthropometric, Renal Characteristics, and Hemodynamics with Aging

Body weight (BW), total kidney weight (TKW), plasma creatinine, GFR aswell as mean arterial pressure (MAP) levels were determined (Table 1).There was a significant increase in BW and TKW at 11 months, which wassustained at 20 months as compared to the 2 month old group. When TKWwas normalized to BW, there was a significant reduction in the TKW:BWratio at 11 and 20 months of age. Further, while plasma creatinine wassignificantly increased at 11 and 20 months compared to 2 month of age,there was a trend for GFR to decrease at 11 months, which was sustainedat 20 months. There was a significant elevation in MAP only at 20 monthsof age.

TABLE 1 Summary of body weight, renal characteristics, and bloodpressure in aging Fischer rats. 2 months 11 months 20 months BW (g) 211± 2  465 ± 5 *  445 ± 7 * 

TKW (mg) 1614 ± 33  2763 ± 73 *  2750 ± 54 *  TKW:BW (mg/g) 7.67 ± 0.195.94 ± 0.15 *  6.18 ± 0.13 * Plasma Cr (mg/dL) 0.20 ± 0.00 0.39 ± 0.03 * 0.36 ± 0.02 * GFR (mL/min/kg) 3.52 ± 0.29 2.61 ± 0.43  2.83 ± 0.30  MAP (mmHg) 90 ± 1  91 ± 2   100 ± 3 * 

BW = body weight; TKW = total kidney weight; Cr = creatinine; GFR =glomerular filtration rate; MAP = mean arterial pressure. Values aremean ± SE. n = 10 for all age groups. * P < 0.05 vs. 2 months,

 P < 0.05 vs. 11 months.

Renal Fibrosis and Glomerular Ultrastructure

Representative photomicrographs of the renal cortex (FIG. 1A) andmedulla (FIG. 2A) stained with picrosirius red were obtained. Thesephotomicrographs provided an estimate of fibrillar collagen deposition.Specifically, there was a significant and progressive increase incortical (FIG. 1B) and medullary (FIG. 2B) interstitial collagenstaining with aging. Further, representative electron photomicrographsof glomeruli were obtained (FIG. 3). At 2 months (FIG. 3A), visceralepithelial cell foot processes were intact. At 11 months (FIG. 3B),mesangial regions were mildly expanded with matrix, and the capillaryloop basement membranes exhibited mild thickening compared to 2 months.At 20 months (FIG. 3C), there was diffuse expansion of mesangial matrix.The capillary loop basement membranes were thickened and exhibited focaleffacement of visceral epithelial cell foot processes as compared 2 and11 months of age. Morphometric analysis of GBM thickness was performed(FIG. 4). There was progressive and significant thickening of capillaryloop basement membranes with aging, where the thickness of basementmembranes at 20 months was almost three times that of the basementmembranes at 2 months of age.

Immunohistochemical Localization of Renal CNP

Immunohistochemical localization of CNP in the renal cortex (left columnpanels) and the medulla (right column panels) was evaluated in Fischerrats at 2, 11, and 20 months of age (FIG. 5). At 2 months of age, therewas no significant staining of proximal tubules, and immunostaining forCNP was localized to distal tubules within the renal cortex. At 11months of age, CNP immunostaining within renal cortex was predominantlylocalized to distal tubules, with faint staining of proximal tubules. At20 months, strong immunostaining for CNP was observed within distaltubules and focally, within proximal tubules of the renal cortex. Strongimmunostaining for CNP was observed within distal tubules of the renalmedulla and did not appreciably change with age.

Additionally, FIG. 10 demonstrates immunohistochemical localization ofCNP in young (A-C) and old (D-F) biopsy specimens from healthy humankidney donors. In biopsies obtained from young kidney donors, CNPstaining was predominantly localized to distal tubules, with relativelyweak, focal staining observed within proximal tubules. In biopsiesobtained from old kidney donors (D-F), strong staining of CNP wasobserved within both distal and proximal tubules.

Urinary and Plasma CNP, Urinary to Plasma CNP Ratio, and Proteinuria

Changes in plasma and urinary CNP with aging were assessed (FIG. 6). Asignificant and progressive decrease in plasma CNP (FIG. 6A) wasobserved between the three age groups (2 month mean±SE: 29±3 pg/mL; 11month mean±SE: 20±1*pg/mL; 20 month mean±SE: 9±1*† pg/mL; *P<0.05 vs. 2months, † P<0.05 vs. 11 months). In contrast, there was a significantincrease in urinary CNP excretion at 11 months, which remained elevatedat 20 months (2 month mean±SE: (64±4 pg/day; 11 month mean±SE:110±6*pg/day; 20 month mean±SE: 103±7*pg/day; *P<0.05 vs. 2 months)(FIG. 6B). Further, there also was a significant and progressiveincrease in the urinary to plasma to CNP ratio (FIG. 6C: 2 monthmean±SE:2.2±0.2 pg/day/pg/mL; 11 month mean±SE:5.4±0.3*pg/day/pg/mL; 20month mean±SE:11.7±1.0*† pg/day/pg/mL; *P<0.05 vs. 2 months, † P<0.05vs. 11 months). A significant increase in proteinuria (FIG. 6D) wasobserved only at 20 months.

Urinary CNP Excretion and Urinary to Plasma CNP Ratio Correlations

A positive correlation was revealed between CNP and renal corticalfibrosis (FIG. 7A; n=21, r=0.54, P=0.01), and between CNP and renalmedullary fibrosis (FIG. 7B; n=21, r=0.65, P=0.001). Further, there wasa strong positive correlation between a urinary to plasma CNP ratio andrenal cortical fibrosis (FIG. 7C; n=21, r=0.83, P=0.0001), and between aurinary to plasma CNP ratio and renal medullary fibrosis (FIG. 7D; n=21,r=0.77, P=0.0001). There was no correlation between urinary CNP and GFR(n=30, r=0.01, P=0.97). FIG. 11 illustrates a strong positivecorrelation among GBM thickness, urinary CNP (FIG. 11A; n=15, r=0.77,P=0.0008) and urinary to plasma CNP ratio (FIG. 11B; n=15, r=0.95,P=0.0001).

The results provided herein demonstrate that urinary CNP excretionincreases during aging and that increased urinary CNP excretion isstrongly associated with renal fibrosis and GBM thickening, whichoccurred prior to the onset of significant proteinuria or BP elevation.The increase in urinary CNP excretion observed with renal aging occurredtogether with a significant increase in the urinary to plasma CNP ratioand decrease in circulating CNP and renal function. These resultsdemonstrate that urinary CNP and its ratio with plasma CNP is abiomarker for early renal structural changes during aging prior to theappearance of clinical signs.

Example 2 CNP is a Urinary Biomarker with Prognostic Value inHospitalized Acute Decompensated Heart Failure (ADHF) PatientsIndependent of Glomerular Filtration Rate and NT-proBNP PatientPopulation

Sixty ADHF (acute decompensated heart failure) patients were studied,and 20 healthy subjects were included as the control group. ADHFpatients were prospectively identified and enrolled from a register ofconsecutive admissions. Inclusion criteria were a clinical diagnosis ofsystolic HF consistent with Framingham criteria (McKee et al., N Engl.J. Med., 285:1441-6 (1971)) for either new onset or established chronicHF, confirmed by reduced (<50%) left ventricular ejection fraction(LVEF) on echocardiography. In order that the study population mayreflect the heterogeneity of normal clinical practice, the onlyexclusion criterion was incomplete or incorrect urine collection foradequate urinary biomarker analysis. Two ADHF patients were excluded forthis reason, leaving a total of 58 consecutive patients providingconsent in the ADHF cohort. All patients underwent baseline historyassessment, physical examination, and transthoracic echocardiography aspart of routine clinical care. Plasma samples for CNP and NT-proBNPmeasurements and 24 hour urine collection were also obtained within 72hours of admission. Urine samples were collected on ice with acetic acid(30 mL of 1:1 acetic acid; 17.4 M). At the end of the timed urinecollection (mean 22.9±4 hours), total volume was recorded, and samplesaliquoted from each container, frozen and stored at −80° C. untilanalysis. For the preliminary analysis, GFR was defined as 24 hourcreatinine clearance. Results were retrospectively verified to concurwith modified diet in renal disease (MDRD) estimates of GFR.

Control subjects were recruited from a population of healthy volunteers.All were non-smokers and had no history of cardiovascular or systemicdisease. Plasma samples for CNP and NT-proBNP measurements and 24-hoururine collections were obtained upon enrollment.

Urine Biomarker Assays NGAL and KIM-1

Urine concentrations of NGAL and KIM-1 were measured by enzyme-linkedimmunoassay as per manufacturer's instructions (Quantikine® ELISA, R&DSystems). The minimum detectable dose for NGAL was 0.012 ng/mL, and theminimum detectable dose for KIM-1 was 0.009 ng/mL. The intra- andinter-assay coefficient of variation for both assays were <5% and <8%,respectively. NGAL is recognized to form complexes with MMP9;recombinant human MMP-9/NGAL complex demonstrated 0.3% cross-reactivityin the assay used. There was no significant cross-reactivity orinterference in the KIM assay.

CNP-22 (AA 82-103)

Urinary CNP-22 was determined by commercially available non-equilibriumradioimmunoassay kits from Phoenix Pharmaceutical (Mountain View,Calif.), using an antibody that detects human CNP-22 as describedelsewhere (Sangaralingham et al., Am. J. Physiol. Renal Physiol.,301:F943-52 (2011)). The range of the standard curve was 0.5-128 pg,with a lower limit of detection of 0.5 pg. Inter- and intra-assayvariability was 11% and 5%, respectively. Recovery was 85%.Cross-reactivity was 0% with ANP, BNP, endothelin, and NT-CNP53, and 59%with CNP-53.

CNP-53 (AA 51-103) and NT-CNP53 (AA 51-81)

Urinary CNP-53 and NT-CNP-53 were determined, similar to that of CNP-22,by commercially available non-equilibrium radioimmunoassay kits fromPhoenix Pharmaceutical (Mountain View, Calif.), using antibodies thatdetect human CNP-53 (CNP-53) and the first 29 amino acids of CNP-53starting from the amino-terminal only when it is separated from the ringstructure (NT-CNP-53). A standard curve was generated and used tocalculate the concentrations of the unknown samples and reported inpg/mL. For CNP-53, the range of the standard curve was 0.5-128 pg.Inter- and intra-assay variability was 8% and 7%, respectively. Recoverywas 81±4%. Cross reactivity was 100% with CNP-22 and 0% with NT-CNP-53,ANP, and BNP. For NT-CNP-53, the range of the standard curve was 0.5-128pg. Inter- and intra-assay variability was 10% and 6%, respectively.Recovery was 82±5.2%. Cross-reactivity was 0% with ANP, BNP, CNP-22,CNP-53, and endothelin.

Urine Biomarker Excretion

Mean urine flow (mL/hour) was determined from total urine volume (mL)and urine collection time (hours). Urine biomarker excretion wascalculated as the product of urine biomarker concentration (pg/mL orng/mL) and urine flow rate (mL/hour) and reported following adjustmentfor urinary creatinine excretion (ng/gCr).

Plasma Biomarker Assays

Blood was drawn into EDTA tubes and chilled until centrifuged at 4° C.,2500 rpm, for 10 minutes. 1 mL plasma was aliquoted and frozen at −20°C. until assayed. Plasma concentrations of CNP molecular forms weredetermined using the same non-equilibrium RIA utilized for urine(Phoenix Pharmaceuticals, Belmont, Calif.); using anti-human CNPantibodies. Plasma NT-proBNP was measured by electrochemiluminescenceimmunoassay as previously described elsewhere (Costello-Boerrigter etal., J. Am. College Cardiol., 47:345-53 (2006)). The lower limit ofdetection for NT-proBNP was 5 pg/mL; inter-assay and intra-assayvariability was 3.1% and 2.5%, respectively. There was nocross-reactivity with CNP forms.

Statistical Analysis

All urinary biomarkers demonstrated a non-Gaussian distribution;therefore, values are presented as median± interquartile range. Forcomparisons between ADHF and control subjects, non-parametric Wilcoxonrank-sum tests were used. Spearman's rank correlation was used toascertain relationships between continuous variables. Biomarkerexcretion data was normalized by natural logarithmic transformationprior to Cox regression analysis to detect independent predictors of:(i) mortality, and (ii) time to first non-elective all-causerehospitalization/death. Mortality and rehospitalization wereascertained from institutional records, which included local primarycare data. Patients were otherwise censored at time of last knownfollow-up. C-statistics were used to compare the discriminatory abilityof biomarkers (Harrell et al., Statistics in medicine, 15:361-87(1996)). The c-statistic is similar to the area under the curve forbinary endpoints and can be interpreted as the probability of correctlyordering event times using risk score from the Cox model. Confidenceintervals were calculated for c-statistics using an approximatejackknife method of calculating standard errors. Additionally, theintegrated discrimination index (IDI) (Pencina et al., Statistics inmedicine, 27:157-72 (2008); discussion 207-12) was utilized to evaluatethe improvement in predictive accuracy using the combination of CNP andplasma NT-proBNP over the use of NT-proBNP alone, for adverse outcomes(mortality and rehospitalization/death) in ADHF. Probability values were2-sided; p<0.05 was considered significant. Data were analyzed using JMPsoftware version 9.0 (SAS Institute, Inc., Cary, N.C.) and SAS version9.2 (SAS Institute, Inc., Cary, N.C.).

Results

Baseline clinical and biochemical characteristics of the studypopulation are shown in Table 2. Patients admitted with ADHF were olderthan controls (70.1±10.3 vs. 53.5±6.1 years; p<0.0001), 23 (40%) werefemale; and mean left ventricular ejection fraction (LVEF) was38.4±18.9%. Twenty two (38%) ADHF patients presented with dyspnea aloneas the predominant symptom; 4 patients (7%) presented with edema alone;and 24 (41%) presented with combined dyspnea and peripheral edema. Theremaining few patients presented with fatigue or ADHF in the context ofarrhythmia-related symptoms. Fifty five percent presented in NYHA ClassIII. Plasma NT-proBNP was significantly elevated in ADHF patients, whileGFR was reduced compared to controls (p<0.0001) (Table 2). Urinarycreatinine concentration was observed to be lower in ADHF than controls(Table 3), likely in accordance with instigation or escalation ofdiuretic therapy during clinical ADHF management.

TABLE 2 Baseline characteristics. Control ADHF p- Variable (n = 20) (n =58) value Age*, y 53.5 ± 6.1 70.1 ± 10.4 <0.0001 Male gender, n (%) 10(50) 35 (59) 0.50 Ischemic etiology, — 19 (33) — n (%) Co-morbidityHypertension, n (%) — 36 (62) — Diabetes, n (%) — 25 (43) — Thyroiddisease, n (%) — 11 (19) — Atrial fibrillation, — 38 (66) — n (%)Previous CVA, n (%) — 7 (12) — CRT, n (%) — 14 (24) — Medications onadmission ACEI or ARB, n (%) — 38 (66) — Beta-blocker, n (%) — 44 (76) —Loop diuretic, n (%) — 49 (84) — Aldosterone — 12 (21) — antagonist, n(%) Mean LVEF*, % — 38.0 ± 18.9 — Serum creatinine*,  0.7 ± 0.18 1.2 ±0.8 <0.0001 mg/dL GFR*, ml/min/1.73 m² 115.9 ± 21.1 60.5 ± 30.3 <0.0001Plasma biomarkers (pg/ml)^(μ) NT-proBNP 37.8 (21.9-7.3) 2461 (1222-6994)<0.0001 CNP-22 6.4 (4.3-18.8) 11.7 (8.3-19.6) 0.005 CNP-53 3.8 (3.6-4.3)5.8 (5.0-7.6) 0.0001 NT-CNP-53 6.5 (5.4-7.7) 6.1 (5.3-6.9) 0.56 *Valuesexpressed as mean (SD) ^(μ)Values expressed as median (25th-75thpercentile). CVA, cerebrovascular accident; CRT, cardiacresynchronization therapy; LVEF, left ventricular ejection fraction;GFR, glomerular filtration rate; NT-proBNP, N-terminal pro-brainnatriuretic peptide; CNP-22, C-type natriuretic peptide-22; CNP-53,C-type natriuretic peptide-53; NT-CNP-53, N-terminal fragment of C-typenatriuretic peptide-53.

Acute Decompensated Heart Failure and Urinary Biomarker Excretion

Excretion rates for all urinary biomarkers displayed a non-Gaussiandistribution. Median excretion of KIM-1 and all three CNP molecularforms was significantly higher in ADHF than controls, as was the urinarytotal protein/creatinine ratio (Table 3 and FIG. 12). Urinary NGALexcretion was unchanged (p=NS). Associations between urinary biomarkerexcretion and clinical characteristics of ADHF patients were explored.KIM-1 demonstrated a weak non-significant association with GFR(Spearman's ρ-0.19; p=0.098), but there were no significantrelationships between any urinary biomarker and NYHA class (III or IV)at presentation, nor any significant trends associated with LVEF (offinotropes) (p=NS for both).

Univariate correlation coefficients between excretion rates of urinaryCNP and other measured HF biomarkers are shown in Table 4. Moderatecorrelations were observed between the three urinary CNP molecular formsbut only urinary CNP-22 displayed any, albeit modest, correlation withits concentration in the plasma (p 0.28, p=0.04). Urinary CNP-22 andCNP-53 were weakly associated with plasma NT-proBNP (p 0.45, p=0.0003; ρ0.33, p=0.01 respectively); urinary NT-CNP-53 was not. Urinary CNP-22 (ρ0.68, p=0.0001) and urinary KIM-1 (ρ 0.78, p<0.0001) demonstrated amarked correlation with urinary total protein/creatinine ratio which wasnot evident with the other urinary biomarkers: CNP-53, NT-CNP-53 orNGAL.

Amongst ADHF patients, medications on admission included angiotensinconverting enzyme inhibitors or angiotensin-receptor blockers (66%), βblockers (76%), loop diuretics (84%), and aldosterone antagonists (21%).On exploratory analysis, urinary NGAL was higher in the context of ACEIor ARB use (median±IQR: 443±1924 vs. 177±227 ng/gCr; p=0.003), andurinary NT-CNP-53 was lower in ADHF patients admitted on loop diuretics(34.0±43.7 vs. 60.4±202.0; p=0.01) than those without. No othersignificant associations were observed between use of these agents onpresentation and urine CNP-22, CNP-53, or KIM-1 levels in the currentcohort.

TABLE 3 Urinary biomarker excretion. Control ADHF p- (n = 20) (n = 58)value Urine 1878.0 (653.7) 1824.8 (1129.3) 0.80 volume* (mL) Urine 24.0(0) 22.9 (4.0) 0.05 collection time* (h) Urinary 75.5 (38.1) 55.3 (37.8)0.04 creati- nine* (mg/dL) Urine 0.02 (0.01-0.02) 0.03 (0.02-0.08)0.0007 protein/ creatinine ratio (mg/mg)^(§) Biomarker excretion(ng/gCr)^(§) KIM-1 475.0 (198.9-604.9) 1354.0 (876.5-2101.5) <0.0001NGAL 298.8 (225.2-458.3) 350.2 (137.2-1405.7) 0.94 CNP-22 7.2 (6.7-9.6)14.0 (8.1-27.0) 0.0003 CNP-53 64.7 (21.6-109.1) 115.2 (63.1-227.8) 0.02NT-CNP- 19.4 (13.3-29.6) 35.8 (20.0-72.6) 0.0015 53 *Values expressed asmean (SD) ^(§)Values expressed as median (25th-75th percentile) KIM-1,kidney injury molecule 1; NGAL, neutrophil gelatinase-associatedlipocalin; CNP-22, C-type natriuretic peptide-22; CNP-53, C-typenatriuretic peptide-53; NT-CNP-53, N-terminal fragment of C-typenatriuretic peptide -53.

TABLE 4 Correlation analysis: Spearman's ρ (rho) rank correlationbetween CNP molecular forms and other potentially important biomarkersof disease severity or prognosis in ADHF patients. Urine Urine CNP-22Urine CNP-53 NT-CNP-53 ρ p-value ρ p-value ρ p-value Age 0.04 0.74 0.080.57 0.06 0.65 GFR −0.11 0.41 0.08 0.53 0.27 0.04 Urine total 0.680.0001 −0.42 0.06 0.04 0.85 protein/ creatinine ratio Urinary biomarkersKIM-1 0.19 0.16 0.30 0.02 0.21 0.11 NGAL 0.43 0.0007 0.12 0.36 0.07 0.61CNP-22 1 — 0.66 <0.0001 0.50 <0.0001 CNP-53 0.66 <0.0001 1 — 0.58<0.0001 NT-CNP-53 0.50 <0.0001 0.58 <0.0001 1 — Plasma biomarkers CNP-220.28 0.04 0.24 0.07 0.26 0.05 CNP-53 0.12 0.39 −0.08 0.52 −0.12 0.38NT-CNP-53 −0.08 0.58 0.09 0.53 −0.14 0.31 NT-proBNP 0.45 0.0003 0.330.01 0.09 0.53 GFR, glomerular filtration rate; KIM-1, kidney injurymolecule 1; NGAL, neutrophil gelatinase-associated lipocalin; CNP-22,C-type natriuretic peptide-22; CNP-53, C-type natriuretic peptide-53;NT-CNP-53, N-terminal fragment of C-type natriuretic peptide -53;NT-proBNP, N-terminal pro-brain natriuretic peptide.

Plasma Concentrations of C-Type Natriuretic Peptide

Plasma concentrations of CNP molecular forms and NT-proBNP are shown inTable 2. Plasma CNP-22 and CNP-53 were elevated in ADHF compared tocontrols, whereas plasma NT-CNP-53 was unchanged. Plasma CNP-22demonstrated limited association to its concurrent urine excretion (ρ0.28, p=0.04), and a weakly positive trend with urine CNP-53 (ρ 0.24,p=0.07) and NT-CNP-53 excretion (ρ 0.26, p=0.05) (Table 4). By contrast,neither plasma CNP-53 nor plasma NT-CNP-53 displayed any relationship tourinary excretion of any CNP molecular form.

Clinical Outcomes

Of the 58 ADHF patients studied, there were 18 deaths (overall ADHFmortality 31%) over a mean (SD) follow-up of 1.5 (0.9) years. Eighteenadditional ADHF patients were rehospitalized (all-causerehospitalization/death rate 62%) of which 13 patients wererehospitalized with a primary presenting complaint of cardiovascularetiology. Two patients were admitted for elective cardiacresynchronization therapy procedures; these were not included as eventsin the final analysis.

ADHF patients who died were older than survivors (74.8±9.2 vs. 67.9±10.2years, p=0.02) but were otherwise similar with respect to gender, NYHAclass, and LVEF. Likewise, ADHF patients who met the secondary outcomeof all-cause rehospitalization/death during follow up were not differentfrom ADHF patients without events in respect to these baselinecharacteristics. Neither plasma NT-proBNP nor GFR were significantlydifferent between ADHF patients with or without adverse outcomes in thiscohort. Of the urinary biomarkers assessed, all three CNP forms wereelevated in ADHF patients who died compared to survivors (FIGS. 13A-Cand 14A-C). Urinary KIM-1 and NGAL excretion were unchanged (p=NS forboth). Plasma CNP-22 was higher in ADHF patients who died than survivors(15.8±18.8 vs. 10.5±8.3 pg/mL; p=0.02), but plasma CNP-53 and NT-CNP-53not significantly different. For patients who met the secondary outcome,urinary CNP-22 and NT-CNP-53 excretion displayed a trend towardselevation (median CNP-22:15.3 vs. 9.3 ng/gCr, p=0.07; NT-CNP-53:37.4 vs.32.6 ng/gCr, p=0.06) as did plasma CNP-22 (13.8±16.3 vs. 10.5±7.7;p=0.06). The remaining urinary biomarkers and plasma CNP forms wereunchanged.

Cox regression analysis (Table 5) revealed only urinary NT-CNP-53excretion to be significantly predictive of mortality (univariate HR1.67, 95% CI 1.14-2.37, p=0.01) and all-cause rehospitalization/death(univariate HR 1.78, 95% CI 1.30-2.39, p=0.0004) from all urinary andplasma biomarkers assessed. Moreover, its association persisted afteradjusting for age, urinary protein/creatinine ratio, and plasmaNT-proBNP (Table 5). On analysis of the c-statistic (c-index) for theoccurrence of all-cause mortality, urinary NT-CNP-53 displayed acomparable c-statistic (0.66; 95% CI 0.53-0.78) to that of NT-proBNP(0.57, 95% CI 0.43-0.71), and the combination of biomarkers, urinaryNT-CNP-53 and plasma NT-proBNP, provided evidence of an incrementaleffect with a combined c-statistic of 0.69 (95% CI 0.56-0.82).Examination of the integrated discrimination index provided furtherevidence that the combination of urinary NT-CNP-53 and plasma NT-proBNPsignificantly improved prediction of adverse outcomes in this cohort(Table 6). No other urinary or plasma biomarker in this studydemonstrated significant predictive value.

TABLE 5 Predictive value of urinary NT-CNP-53 excretion and plasmaNT-proBNP for clinical outcome in ADHF patients. Univariate and adjustedCox proportional hazard analysis. Outcome All-cause Deathrehospitalization/death Model HR (95% CI) p-value HR (95% CI) p-valueUrinary NT- CNP53* Unadjusted 1.67 (1.14-2.37) 0.01 1.78 (1.30-2.39)0.0004 Model 1 1.54 (1.05-2.22) 0.03 1.75 (1.28-2.36) 0.0007 Model 21.60 (1.06-2.38) 0.03 1.74 (1.26-2.36) 0.001 Model 3 1.67 (1.08-2.57)0.02 1.79 (1.28-2.47) 0.0009 Plasma NT- proBNP* Unadjusted 1.24(0.83-1.85) NS 1.21 (0.92-1.60) NS Model 1 1.26 (0.83-1.87) NS 1.22(0.93-1.61) NS Model 2 1.30 (0.85-1.98) NS 1.22 (0.91-1.63) NS *Lntransformed data (hazard ratio are per 1 log unit increase) Model 1:Adjusted for age Model 2: Adjusted for age and urine protein/creatinineratio Model 3: Adjusted for age, urine protein/creatinine ratio, andplasma NT-proBNP CV, cardiovascular; NT-CNP-53, N-terminal fragment ofC-type natriuretic peptide -53; NT-proBNP, N-terminal pro-B-typenatriuretic peptide; NS, not significant (i.e. p > 0.05).

TABLE 6 Measures of predictive accuracy. Integrated discriminationimprovement, Model C-index (95% CI) % (SE) p-value Death NT-proBNP^(†)0.57 (0.43-0.71) — — NT-CNP-53^(‡) 0.66 (0.53-0.78) — — NT-proBNP^(†)and 0.69 (0.56-0.82) 30 (11)*  0.004* NT-CNP-53^(‡)Death/rehospitalization NT-proBNP^(†) 0.56 (0.46-0.66) — — NT-CNP-53^(‡)0.67 (0.59-0.76) — NT-proBNP^(†) and 0.69 (0.61-0.78) 17 (5.0)* 0.001*NT-CNP-53^(‡) ^(†)Plasma NT-proBNP ^(‡)Urinary NT-CNP-53 *compared toNT-proBNP alone NT-proBNP, N-terminal pro-B type natriuretic peptide;NT-CNP-53, N-terminal fragment of C-type natriuretic peptide -53; SE,standard error.

These results demonstrate that elevated KIM-1 excretion appears todiscriminate between decompensated heart failure patients and healthycontrol patients, but does not correlate with heart failure outcome. Bycontrast, elevated urinary NT-CNP-53 excretion demonstrated asignificant correlation with adverse outcome within a heterogeneoushospitalized heart failure population (e.g., ADHF patients), independentof GFR. NT-CNP-53 was the only urinary biomarker with predictive value.

Example 3 Plasma CNP-22 is an Endothelial Cell Biomarker that PredictsMortality and Myocardial Infarction in the General Population

The following was performed to determine if plasma CNP-22 is anendothelial cell derived biomarker for predicting future mortality andmyocardial infarction (MI) in the general population. Plasma CNP-22 wasassessed in 1,841 subjects (mean age 62±11 years, 48% male) randomlyselected from the general community of Olmsted County, MN, USA. Medianfollow-up for mortality and MI was 12 years. Over the 12 year follow-upperiod, elevated plasma CNP-22 (CNP-22>16 pg/mL) was significantlyassociated with mortality (unadjusted HR 1.41, 95% CI 1.12-1.79;P=0.004) and MI (unadjusted HR 1.60, 95% CI 1.19-2.16; P=0.002) (Table7). After adjusting for traditional risk factors (e.g., age, gender,body mass index (BMI), cholesterol, serum creatinine, smoking, andpresence of diabetes and hypertension), elevated plasma CNP-22 levelsremained significantly associated with mortality (adjusted HR 1.34, 95%CI 1.02-1.75; P=0.04) and MI (adjusted HR 1.59, 95% CI 1.13-2.25;P=0.008) (Table 7).

TABLE 7 Elevated plasma CNP-22 levels predict mortality andcardiovascular morbidity (overall cohort: n = 1841). Outcome HR (95% CI)Log CNP-22 p Value Death (n = 328) Unadjusted 1.414 (1.115-1.793) 0.0043Age, Sex, BMI 1.326 (1.016-1.730) 0.0380 Model 3 1.336 (1.019-1.752)0.0362 MI (n = 189) Unadjusted 1.602 (1.187-2.161) 0.0021 Age, Sex, BMI1.699 (1.219-2.369) 0.0018 Model 3 1.591 (1.128-2.245) 0.0082 Model 3:age, sex, BMI, total cholesterol, serum creatinine, smoking, presence ofdiabetes, hypertension, coronary artery disease

Death and MI according to quartiles of plasma CNP-22 are shown in FIGS.15A and 15B, respectively. The unadjusted incidence of death and MIevents significantly increased with increasing quartiles of plasmaCNP-22, where CNP-22 Q1=2.0 to 10.1 pg/mL; CNP-22 Q2=10.2 to 13.1 pg/mL;CNP-22 Q3=13.2 to 16.7 pg/mL; and CNP-22 Q4=16.8 to 265.0 pg/mL.

These results demonstrate that an elevated plasma CNP-22 level is anendothelial cell biomarker that can predict future cardiac-related deathand MI in the general community. These results also demonstrate thathumans with elevated plasma CNP-22 levels can be subjected to early MIdetection strategies and/or aggressive therapeutic strategies for MIprevention.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-60. (canceled)
 61. A method for treating a mammal having an increasedrisk of a renal structural alteration, wherein said method comprises:(a) determining that said mammal has an elevated urinary CNP to plasmaCNP ratio, (b) monitoring said mammal for the presence of a risk factorfor renal structural alteration, and (c) instructing said mammal toadminister a therapeutic agent to reduce a symptom of said renalstructural alteration.
 62. The method of claim 61, wherein said mammalis a human.
 63. The method of claim 61, wherein said elevated urinaryCNP to plasma CNP ratio is greater than 12,000 pg/day to 16 pg/mL. 64.The method of claim 61, wherein said elevated urinary CNP to plasma CNPratio is greater than 13,200 pg/day to 18 pg/mL.
 65. The method of claim61, wherein said risk factor is selected from the group consisting of anage factor, hypertension, an elevated serum creatinine level,proteinuria, an elevated body mass index, an elevated cholesterol level,a smoking habit, and diabetes.
 66. The method of claim 61, wherein saidtherapeutic agent is an ACE inhibitor, an angiotensin receptor blocker,an aldosterone antagonist, a statin, a native natriuretic peptide, or adesigner natriuretic peptide.
 67. A method for treating a mammal havingan increased risk of a poor outcome, wherein said method comprises: (a)determining that said mammal has an elevated level of urinary CNP, (b)monitoring said mammal for the presence of a risk factor for a pooroutcome, and (c) instructing said mammal to administer a therapeuticagent to reduce the likelihood of said poor outcome.
 68. The method ofclaim 67, wherein said mammal is a human.
 69. The method of claim 67,wherein said urinary CNP is urinary NT-CNP-53.
 70. The method of claim67, wherein said elevated level is greater than 36,000 pg ofNT-CNP-53/day.
 71. The method of claim 67, wherein said elevated levelis greater than 42,000 pg of NT-CNP-53/day.
 72. The method of claim 67,wherein said risk factor is selected from the group consisting of an agefactor, hypertension, an elevated serum creatinine level, proteinuria,an elevated body mass index, an elevated cholesterol level, a smokinghabit, and diabetes.
 73. The method of claim 67, wherein saidtherapeutic agent is an ACE inhibitor, an angiotensin receptor blocker,an aldosterone antagonist, a statin, a native natriuretic peptide, or adesigner natriuretic peptide.
 74. A method for treating a mammal havingan increased risk of myocardial infarction, wherein said methodcomprises: (a) determining that said mammal has an elevated level ofplasma CNP-22, (b) monitoring said mammal for the presence of a riskfactor for myocardial infarction, and (c) instructing said mammal toadminister a therapeutic agent to reduce the risk of myocardialinfarction.
 75. The method of claim 74, wherein said mammal is a human.76. The method of claim 74, wherein said elevated level is greater than14 pg/mL.
 77. The method of claim 74, wherein said elevated level isgreater than 16 pg/mL.
 78. The method of claim 74, wherein said riskfactor is selected from the group consisting of an age factor,hypertension, an elevated serum creatinine level, proteinuria, anelevated body mass index, an elevated cholesterol level, a smokinghabit, and diabetes.
 79. The method of claim 74, wherein saidtherapeutic agent is an ACE inhibitor, an angiotensin receptor blocker,an aldosterone antagonist, a statin, a native natriuretic peptide, or adesigner natriuretic peptide.